The following references may contribute to the understanding of the invention, and are referred to by number in the specification:    1. Zuker A, Tzfira T, Vainstein A: Genetic engineering for cut-flower improvement. Biotech Adv 16: 33–79 (1998)    2. van Altvorst A C, Rikesen T, Koehorst H, Dons J J M: Transgenic carnations obtained by Agrobacterium tumefaciens-mediated of leaf explants. Transgenic Res 4:105–113 (1995)    3. Zuker A, Chang P-F L, Ahroni A, Cheah K, Woodson W R, Bressan R A, Watad A A, Hasegawa P M, Vainstein A: Transformation of carnation by microprojectile bombardment. Sci Hort 64: 177–185 (1995)    4. Firoozabady E, Moy Y, Tucker W, Robinson K, Gutterson N: Efficient transformation and regeneration of carnation cultivars using Agrobacterium. Molecular Breeding 1: 283–293 (1995)    5. Lu C Y, Nugent G, Wardley-Richardson T, Chandler S F, Young R, Dalling M J: Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.). Bio/Tech 9: 864–868 (1991)    6. Zuker, A., et al: Application of an integrative system based on microprojectile bombardment and Agrobacterium tumefaciens to generate transgenic carnation plants with novel characteristics. IX International Congress on Plant Tissue and Cell Culture, Jerusalem, Israel (1998)    7. Fladung, M., K. Grossmann, and M. R. Ahuja. 1997. Alterations in hormonal and developmental characteristics in transgenic Populus conditioned by the rolC gene from Agrobacterium rhizogenes. J. Plant Physiol. 150:420–427    8. Scorza, R., T. W. Zimmerman, J. M. Cordts, K. J. Footen, and M. Ravelonandro. 1994. Horticultural characteristics of transgenic tobacco expressing rolC gene from Agrobacterium rhizogenes. J. Amer. Soc. Hort. Sci. 119:1091–1098    9. Nilsson, O. and O. Olsson. 1997. Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant. 100:463–473    10. Souq, F., P. Coutos-Thevenot, H. Yean, G. Delbard, Y Maziere, J. P. Barbe, and M. Boulay. 1986. Genetic transformation of roses, 2 examples: one on morphogenesis, the other on anthocyanin biosynthetic pathway. In: Morisot A, Ricci P (eds) Second International Symposium on Roses. Acta Hort. 424:381–388    11. Pelletier, M. K. & Shirley, B. W. Analysis of flavanone 3-hydroxylase in arabidopsis seedlings. Plant Physiol. 111, 339–345 (1996)    12. Ahroni A: Developing efficient regeneration and transformation methods for carnation and gypsophila. M. Sc. thesis (The Hebrew University of Jerusalem, Israel) (1996)    13. Tzfira, T., C. S. Jensen, W. Wang, A. Zuker, B. Vinocur, A. Altman, and A. Vainstein. 1997. Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 15:219–235
The carnation (Dianthus L.), one of the world's major cut-flower crops, is the commercial leader in terms of number of stems sold worldwide. Different carnation types have been developed as a result of crossing and they can be divided into two main groups, standard (midi and mignon carnation) and spray (micro and diantini carnation); a minor group is formed by the pot carnations.
Market demand for flowers with improved traits—such as new colors, new shapes, better fragrance and longer vase life, drives breeders to create new and more attractive varieties every year. However, carnation is a vegetatively propagated crop and is very detrimentally affected by inbreeding. Hence, controlled breeding is rather complicated and limited due to the fact that selection of a desired trait in the siblings is performed on the genetic background of their two parents, and due to the very high genetic variability among offspring. Furthermore, crosses within and between related species is limited by a rather small available gene pool for new traits.
Biotechnological techniques such as genetic engineering could be a useful alternative/addition to currently used classical breeding methods for the production of novel plant varieties (1).
One of the prerequisites to generating transgenic plants is the availability of a regeneration system. For carnation, numerous procedures leading to efficient adventitious regeneration from a number of explants, including stem segments, leaves and petals, have been published (1). However, success in harnessing these regeneration systems to efficiently generate transgenes has been rather limited. The Agrobacterium-mediated transformation procedure described by van Altvorst et al. (2) and the microprojectile bombardment-mediated procedure (3) were both of relatively low efficiency.
A more recent, alternative transformation procedure was developed by DNAP Inc. (4). Although a high number of transgenes could be generated from the three varieties used, the procedure was extremely time-consuming and cumbersome because it relied on vitrified leaves as the primary explants and these can take 4 to 6 months of tissue culture to generate. The only efficient procedure reported to date, also used to generate transgenes with improved vase life, was developed by another biotech company, Calgene Pacific Ltd. (5). However extensive efforts by numerous groups to generate carnation transgenes with this experimental protocol have been unsuccessful. A preliminary report of an integrative system based on microprojectile bombardment and Agrobacterium-mediated transformation under a light regime and a two-step selection cycle has recently appeared (6).
New traits in cut flowers include not only yield improvement and resistance to insects or disease; they also consist of new colors and novel plant morphology. In fact, the latter are of great importance for the cut-flower market, where plant architecture and flower color are the main features determining consumer interest. Nevertheless, agronomic traits, such as performance, remain highly important for breeders and growers as extensively growing and better performing plants can lower the time and cost required for growth and breeding.
Among the different genes affecting plant morphology, e.g. homeotic, Agrobacterium, phytochrome and gibberellin genes, the Agrobacterium rhizogenes rol genes have been the most widely and successfully employed (7,8). Although its precise mode of action is still unknown (9), rolC has attracted the most attention through its expression in transgenic plant—either under its own promoter or under the control of a cauliflower mosaic virus (CaMV) 35S promoter, leading to a series of morphological alterations. These include reduced apical dominance, altered leaf morphology, reduced seed production, reduced internode length, male sterility, small flowers and early flowering, bushy and compact phenotype, and even stem fasciation.
Nevertheless, studies on the rolC gene and its effects on plant development have been performed mostly in model herbaceous plants, e.g. tobacco, potato, and tomato, or forest trees. rolC studies in cut flowers are rather limited—the only report being on a woody ornamental, rose, in the proceedings of a symposium (10). These transgenic rose plants (Rosa hybrida cv. Madame G. Delbard), expressing the rolC gene under its native promoter, exhibited an array of phenotypic alterations, most of which were highly disadvantageous horticulturally. They included a dramatically reduced root system, wrinkled leaves, high sensitivity to diseases and dying out of shoots.
With respect to color, anthocyanins, carotenoids and betalain are the main flower pigments, of which the most studied are the anthocyanins. The great interest in the latter, which derive from the general phenylpropanoid pathway, results from their wide distribution in many plants and microorganisms and their role in color determination. Furthermore, detailed biochemical and genetic analyses of anthocyanin production/accumulation has brought about the development of two main strategies for altering flower color: introducing a foreign gene(s) to allow new branching in the anthocyanin-biosynthesis pathway, and up/down-regulation of this pathway's native genes' expression.
Several genes from the anthocyanin biosynthetic pathway have been used to manipulate flower color in numerous plants. Chalcone synthase (chs), chalcone flavanone isomerase (chi) and flavanone 3-hydroxylase (fht) are the first three genes encoding the early, non-branched segment of the flavonoid biosynthetic pathway (FIG. 17). Although FHT is a key enzyme in this pathway (11) and its suppression, as opposed to that of CHS, should not block the production of the essential phytoalexins, isoflavonoids, it has never been harnessed for the genetic manipulation of flower color.
With respect to flower fragrance, mainly dominated by terpenoids, fatty acid derivatives and benzenoid compounds (phenylpropanoid derivatives), much less is known. To date, only a few genes involved in fragrance production have been characterized.